Anti-VEGF antibodies

ABSTRACT

The invention herein provides isolated antibodies that bind to VEGF. The invention further provides methods of making anti-VEGF antibodies, and polynucleotides encoding anti-VEGF antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/039,881, filed Mar. 3, 2011, now U.S. Pat. No. 8,921,537, which is acontinuation of U.S. application Ser. No. 12/315,220, filed Dec. 1,2008, now U.S. Pat. No. 7,910,098, which claims the benefit of thefiling date of U.S. provisional application No. 60/991,302, filed Nov.30, 2007, herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to anti-VEGF selected polypeptidesequences and antibodies with beneficial properties for research,therapeutic and diagnostic purposes.

BACKGROUND OF THE INVENTION

Development of a vascular system is a fundamental requirement for manyphysiological and pathological processes. Actively growing tissues suchas embryos and tumors require adequate blood supply. They satisfy thisneed by producing pro-angiogenic factors, which promote new blood vesselformation via a process called angiogenesis. Vascular tube formation isa complex but orderly biological event involving all or many of thefollowing steps: a) Endothelial cells (ECs) proliferate from existingECs or differentiate from progenitor cells; b) ECs migrate and coalesceto form cord-like structures; c) vascular cords then undergotubulogenesis to form vessels with a central lumen; d) existing cords orvessels send out sprouts to form secondary vessels; e) primitivevascular plexus undergo further remodeling and reshaping; and f)peri-endothelial cells are recruited to encase the endothelial tubes,providing maintenance and modulatory functions to the vessels; suchcells including pericytes for small capillaries, smooth muscle cells forlarger vessels, and myocardial cells in the heart. Hanahan, D. Science277:48-50 (1997); Hogan, B. L. & Kolodziej, P. A. Nature ReviewsGenetics. 3:513-23 (2002); Lubarsky, B. & Krasnow, M. A. Cell. 112:19-28(2003).

It is well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors andmetastasis, atherosclerosis, retrolental fibroplasia, hemangiomas,chronic inflammation, intraocular neovascular diseases such asproliferative retinopathies, e.g., diabetic retinopathy, age-relatedmacular degeneration (AMD), neovascular glaucoma, immune rejection oftransplanted corneal tissue and other tissues, rheumatoid arthritis, andpsoriasis. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992);Klagsbrun et al., Annu. Rev. Physiol. 53:217-239 (1991); and Garner A.,“Vascular diseases,” In: Pathobiology of Ocular Disease. A DynamicApproach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker,NY, 1994), pp 1625-1710.

In the case of tumor growth, angiogenesis appears to be crucial for thetransition from hyperplasia to neoplasia, and for providing nourishmentfor the growth and metastasis of the tumor. Folkman et al., Nature339:58 (1989). The neovascularization allows the tumor cells to acquirea growth advantage and proliferative autonomy compared to the normalcells. A tumor usually begins as a single aberrant cell, which canproliferate only to a size of a few cubic millimeters due to thedistance from available capillary beds, and it can stay ‘dormant’without further growth and dissemination for a long period of time. Sometumor cells then switch to the angiogenic phenotype to activateendothelial cells, which proliferate and mature into new capillary bloodvessels. These newly formed blood vessels not only allow for continuedgrowth of the primary tumor, but also for the dissemination andrecolonization of metastatic tumor cells. Accordingly, a correlation hasbeen observed between density of microvessels in tumor sections andpatient survival in breast cancer as well as in several other tumors.Weidner et al., N. Engl. J. Med 324:1-6 (1991); Horak et al., Lancet340:1120-1124 (1992); Macchiarini et al., Lancet 340:145-146 (1992). Theprecise mechanisms that control the angiogenic switch is not wellunderstood, but it is believed that neovascularization of tumor massresults from the net balance of a multitude of angiogenesis stimulatorsand inhibitors (Folkman, Nat Med 1(1):27-31 (1995)).

The process of vascular development is tightly regulated. To date, asignificant number of molecules, mostly secreted factors produced bysurrounding cells, have been shown to regulate EC differentiation,proliferation, migration and coalescence into cord-like structures. Forexample, vascular endothelial growth factor (VEGF) has been identifiedas the key factor involved in stimulating angiogenesis and in inducingvascular permeability. Ferrara et al., Endocr. Rev. 18:4-25 (1997). Thefinding that the loss of even a single VEGF allele results in embryoniclethality points to an irreplaceable role played by this factor in thedevelopment and differentiation of the vascular system. Furthermore,VEGF has been shown to be a key mediator of neovascularizationassociated with tumors and intraocular disorders. Ferrara et al.,Endocr. Rev. supra. The VEGF mRNA is overexpressed by the majority ofhuman tumors examined. Berkman et al., J. Clin. Invest. 91:153-159(1993); Brown et al., Human Pathol. 26:86-91 (1995); Brown et al.,Cancer Res. 53:4727-4735 (1993); Mattern et al., Brit. J. Cancer73:931-934 (1996); Dvorak et al., Am. J. Pathol. 146:1029-1039 (1995).

Also, the concentration levels of VEGF in eye fluids are highlycorrelated to the presence of active proliferation of blood vessels inpatients with diabetic and other ischemia-related retinopathies. Aielloet al., N. Engl. J. Med. 331:1480-1487 (1994). Furthermore, studies havedemonstrated the localization of VEGF in choroidal neovascular membranesin patients affected by AMD. Lopez et al., Invest. Ophthalmol. Vis. Sci.37:855-868 (1996).

Anti-VEGF neutralizing antibodies suppress the growth of a variety ofhuman tumor cell lines in nude mice (Kim et al., Nature 362:841-844(1993); Warren et al., J. Clin. Invest. 95:1789-1797 (1995); Borgströmet al., Cancer Res. 56:4032-4039 (1996); Melnyk et al., Cancer Res.56:921-924 (1996)) and also inhibit intraocular angiogenesis in modelsof ischemic retinal disorders. Adamis et al., Arch. Ophthalmol.114:66-71 (1996). Therefore, anti-VEGF monoclonal antibodies or otherinhibitors of VEGF action are promising candidates for the treatment oftumors and various intraocular neovascular disorders. Such antibodiesare described, for example, in EP 817,648 published Jan. 14, 1998; andin WO98/45331 and WO98/45332, both published Oct. 15, 1998. One of theanti-VEGF antibodies, bevacizumab, has been approved by the FDA for usein combination with a chemotherapy regimen to treat metastaticcolorectal cancer (CRC) and non-small cell lung cancer (NSCLC). Andbevacizumab is being investigated in many ongoing clinical trials fortreating various cancer indications.

Other anti-VEGF antibodies, anti-Nrp1 antibodies and anti-Nrp2antibodies are also known, and described, for example, in Liang et al.,J Mol Biol 366, 815-829 (2007) and Liang et al., J Biol Chem 281,951-961 (2006), PCT publication number WO2007/056470 and PCT ApplicationNo. PCT/US2007/069179, the content of these patent applications areexpressly incorporated herein by reference.

SUMMARY OF THE INVENTION

The invention provides novel anti-VEGF antibodies and uses thereof.

A number of anti-VEGF antibodies are provided in the invention. Forexample, an antibody that binds to VEGF or a fragment thereof isprovided, wherein the antibody comprises six HVRs selected from:

-   -   (i) an HVR-L1 comprising the amino acid sequence of X₁X₂R X₃SL        wherein the HVR-L1 comprises 1, 2 or 3 substitutions in any        combination of the following positions: X₁ is G or A; X₂ is V or        I; and/or X₃ is T or R;    -   (ii) an HVR-L2 comprising the amino acid sequence of DASSLA (SEQ        ID NO:6);    -   (iii) an HVR-L3 comprising the amino acid sequence of SYKSPL        (SEQ ID NO:7);    -   (iv) an HVR-H1 comprising the amino acid sequence of SISGSWIF        (SEQ ID NO:1);    -   (v) an HVR-H2 comprising the amino acid sequence of GAIWPFGGYTH        (SEQ ID NO:2); and    -   (vi) an HVR-H3 comprising the amino acid sequence of        RWGHSTSPWAMDY (SEQ ID NO:3).

In another embodiment, an antibody that binds to VEGF or a fragmentthereof is provided, wherein the antibody comprises:

-   -   (1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2;    -   (3) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3    -   (4) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;    -   (5) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;        and    -   (6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In another embodiment, an antibody that binds to VEGF or a fragmentthereof is provided, wherein the antibody comprises:

-   -   (1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2;    -   (3) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3    -   (4) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;    -   (5) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;        and    -   (6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In another embodiment, an antibody that binds to VEGF or a fragmentthereof is provided, wherein the light chain variable domain comprisesthe amino acid sequence of SEQ ID NO:44 or SEQ ID NO:45.

In another embodiment, an antibody that binds to VEGF or a fragmentthereof is provided, wherein the anti-VEGF antibody comprises the heavychain variable domain comprising the amino acid sequence of SEQ IDNO:43, and the light chain variable domain comprises the amino acidsequence of SEQ ID NO:44 or 45. In yet another embodiment, the anti-VEGFantibody comprises the heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO:43, and the light chain variable domaincomprises the amino acid sequence of SEQ ID NO:44. In yet anotherembodiment, the anti-VEGF antibody comprises the heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:43, and the lightchain variable domain comprises the amino acid sequence of SEQ ID NO:45.

In certain embodiments, any of the above antibodies is a monoclonalantibody. In one embodiment, the antibody is an antibody fragmentselected from a Fab, Fab′-SH, Fv, scFv, or (Fab′)₂ fragment. In oneembodiment, the antibody is humanized. In one embodiment, the antibodyis human. In yet another embodiment, at least a portion of the frameworksequence is a human consensus framework sequence.

Polynucleotides encoding any of the above antibodies are provided, aswell as vectors comprising the polynucleotides and host cells comprisingthe vectors of the invention. In one embodiment, the host cell iseukaryotic. In another embodiment, the host cell is a CHO cell. A methodof making an anti-VEGF antibody is also provided. For example, a methodcomprises culturing the host cell under conditions suitable forexpression of the polynucleotide encoding the antibody, and isolatingthe antibody.

In one aspect, a method of detecting the presence of VEGF in abiological sample is provided, the method comprising contacting thebiological sample with an antibody of the invention under conditionspermissive for binding of the antibody to VEGF, and detecting whether acomplex is formed between the antibody and VEGF. In one embodiment, themethod comprises detecting VEGF-anti-VEGF antibody complex in abiological sample wherein the amino acid sequence of the anti-VEGFantibody comprises a heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO:43, and a light chain variable domaincomprising the amino acid sequence of SEQ ID NO:44 or 45. In yet anotherembodiment, the method comprises detecting VEGF-anti-VEGF antibodycomplex in a biological sample wherein the amino acid sequence of theanti-VEGF antibody comprises a heavy chain variable domain comprisingthe amino acid sequence of SEQ ID NO:43, and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO:44. In yetanother embodiment, the method comprises detecting VEGF-anti-VEGFantibody complex in a biological sample wherein the amino acid sequenceof the anti-VEGF antibody comprises a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:43, and a light chainvariable domain comprising the amino acid sequence of SEQ ID NO:45. Inyet another embodiment, the anti-VEGF antibody is detectably labeled.

Pharmaceutical compositions as well as methods of treatment are alsoprovided. In one aspect, a pharmaceutical composition is providedcomprising an antibody of the invention and a pharmaceuticallyacceptable carrier. In another aspect, a method of treating cancer isprovided, e.g., the method comprises administering to an individual thepharmaceutical composition comprising any of the above antibodies.Cancers treated by the methods of the invention include, but are notlimited to, squamous cell cancer, small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastric cancer,gastrointestinal cancer, gastrointestinal stromal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer, melanoma, superficial spreading melanoma, lentigo malignamelanoma, acral lentiginous melanomas, nodular melanomas, B-celllymphoma, chronic lymphocytic leukemia (CLL); acute lymphoblasticleukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia;post-transplant lymphoproliferative disorder (PTLD), abnormal vascularproliferation associated with phakomatoses, edema associated with braintumors, or Meigs' syndrome. In certain embodiments, the tumor, cancer orcell proliferative disorder being treated colon cancer, lung cancer,breast cancer or glioblastoma. In yet another embodiment, the subjectbeing treated is human.

The invention further provides immunoconjugates comprising an antibodyconjugated to an agent, such as a drug or cytotoxic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Heavy chain and light chain HVR loop sequences of anti-VEGFantibodies. The figures show the heavy chain HVR sequences, H1, H2, andH3, and light chain HVR sequences, L1, L2 and L3. Sequence numbering isas follows: clone B20-4.1.1 (HVR-H1 is SEQ ID NO:1; HVR-H2 is SEQ IDNO:2; HVR-H3 is SEQ ID NO:3; HVR-L1 is SEQ ID NO:4; HVR-L2 is SEQ IDNO:6; HVR-L3 is SEQ ID NO:7); and clone B20-4.1.1RR (HVR-H1 is SEQ IDNO:1; HVR-H2 is SEQ ID NO:2; HVR-H3 is SEQ ID NO:3; HVR-L1 is SEQ IDNO:5; HVR-L2 is SEQ ID NO:6; HVR-L3 is SEQ ID NO:7).

Amino acid positions are numbered according to the Kabat numberingsystem as described below.

FIGS. 2a and 2b : depict exemplary acceptor human consensus frameworksequences for use in practicing the instant invention with sequenceidentifiers as follows:

Variable Heavy (VH) Consensus Frameworks

-   -   human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID        NO:8)    -   human VH subgroup I consensus framework minus extended        hypervariable regions (SEQ ID NOs:9-11)    -   human VH subgroup II consensus framework minus Kabat CDRs (SEQ        ID NO:12)    -   human VH subgroup II consensus framework minus extended        hypervariable regions (SEQ ID NOs:13-15)    -   human VH subgroup II consensus framework minus extended    -   human VH subgroup III consensus framework minus Kabat CDRs (SEQ        ID NO:16)    -   human VH subgroup III consensus framework minus extended        hypervariable regions (SEQ ID NOs:17-19)    -   human VH acceptor framework minus Kabat CDRs (SEQ ID NO:20)    -   human VH acceptor framework minus extended hypervariable regions        (SEQ ID NOs:21-22)    -   human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO:23)    -   human VH acceptor 2 framework minus extended hypervariable        regions (SEQ ID NOs:24-26)

Amino acid positions are numbered according to the Kabat numberingsystem as described below.

FIG. 3: depicts exemplary acceptor human consensus framework sequencesfor use in practicing the instant invention with sequence identifiers asfollows:

Variable Light (VL) Consensus Frameworks

-   -   human VL kappa subgroup I consensus framework (SEQ ID NO:27)    -   human VL kappa subgroup II consensus framework (SEQ ID NO:28)    -   human VL kappa subgroup III consensus framework (SEQ ID NO:29)    -   human VL kappa subgroup IV consensus framework (SEQ ID NO:30)

FIG. 4: depicts framework region sequences of huMAb4D5-8 heavy and lightchains. Numbers in superscript/bold indicate amino acid positionsaccording to Kabat.

FIG. 5: depicts modified/variant framework region sequences ofhuMAb4D5-8 heavy and light chains. Numbers in superscript/bold indicateamino acid positions according to Kabat.

FIG. 6: The amino acid sequences of the light chain HVR sequences, L1,L2 and L3 for anti-VEGF antibodies B20-4.1.1 and B20-4.1.1RR.

FIG. 7: The amino acid sequences of the heavy chain HVR sequences, H1,H2, and H3 for anti-VEGF antibodies B20-4.1.1 and B20-4.1.1RR.

FIG. 8: depicts the light chain variable regions of antibody clonesB20-4.1.1 and B20-4.1.1RR.

FIG. 9: depicts the heavy chain variable regions of antibody clonesB20-4.1.1 and B20-4.1.1RR.

FIG. 10: Table summarizing the kinetic binding affinity measurement ofB20 variants IgGs to human VEGF and murine VEGF. Human or murine VEGFwas immobilized approximately 60 response unit.

FIG. 11: Table summarizing the kinetic binding affinity measurement ofB20 variants IgGs to human VEGF and murine VEGF. Human or murine VEGFwas immobilized approximately 1000 response unit.

FIG. 12: HUVEC thymidine incorporation assay, which shows that the B20variants can effectively inhibit the HUVEC cell proliferation.

FIG. 13: depicts the effect of B20-4.1.1 on VEGF-induced BRMEproliferation.

FIG. 14: depicts the effect of B20-4.1.1 on tumor growth in nude micewith xenografted human tumor cells (A549 cells), as measured by tumorvolumes over the number of treatment days.

FIG. 15: depicts the effect of B20-4.1.1 on tumor growth in nude micewith xenografted human tumor cells (MDA-MB231 cells), as measured bytumor volumes over the number of treatment days.

FIG. 16: depicts the effect of the avastin antibody on VEGF-induced BRMEproliferation. Inhibition of mVEGF was not observed at a concentrationof up to 1500 nM of the avastin antibody.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein provides isolated antibodies that bind to VEGF anduses thereof. Pharmaceutical compositions as well as methods oftreatment are also provided.

The invention further provides methods of making anti-VEGF antibodies,and polynucleotides encoding anti-VEGF antibodies.

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993).

Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and, whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with any document incorporatedherein by reference, the definition set forth below shall control.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo etal., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132(2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen(i.e., has a binding affinity (K_(d)) value of no more than about 1×10⁻⁷M, preferably no more than about 1×10⁻⁸ M and most preferably no morethan about 1×10⁻⁹ M), but has a binding affinity for a homologue of theantigen from a second nonhuman mammalian species which is at least about50 fold, or at least about 500 fold, or at least about 1000 fold, weakerthan its binding affinity for the human antigen. The species-dependentantibody can be any of the various types of antibodies as defined above,but preferably is a humanized or human antibody.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Inone embodiment, the antibody mutant will have an amino acid sequencehaving at least 75% amino acid sequence identity or similarity with theamino acid sequence of either the heavy or light chain variable domainof the species-dependent antibody, in another embodiment at least 80%,in another embodiment at least 85%, in another embodiment at least 90%,and yet in another embodiment at least 95%. Identity or similarity withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical (i.e, sameresidue) or similar (i.e., amino acid residue from the same group basedon common side-chain properties, see below) with the species-dependentantibody residues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibodies includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al., Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1,L2, L3). In native antibodies, H3 and L3 display the most diversity ofthe six HVRs, and H3 in particular is believed to play a unique role inconferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g., residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see U.S. Provisional Application No. 60/640,323, Figures for EUnumbering).

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies may be produced using certainprocedures known in the art. For example, Marks et al., Bio/Technology10:779-783 (1992) describes affinity maturation by VH and VL domainshuffling. Random mutagenesis of HVR and/or framework residues isdescribed by, for example, Barbas et al., Proc Nat. Acad. Sci. USA91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton etal., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896(1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which partially orfully mimics at least one of the functional activities of a polypeptideof interest.

“Growth inhibitory” antibodies are those that prevent or reduceproliferation of a cell expressing an antigen to which the antibodybinds.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays as disclosed, for example, in definitions herein.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc region,as well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g., from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daëron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995)). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie etal., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol.Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

Binding to human FcRn in vivo and serum half life of human FcRn highaffinity binding polypeptides can be assayed, e.g., in transgenic miceor transfected human cell lines expressing human FcRn, or in primates towhich the polypeptides with a variant Fc region are administered. WO2000/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al., J. Biol.Chem. 9(2):6591-6604 (2001).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils. The effector cells may be isolated from a native source,e.g., from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., NK cells, neutrophils, andmacrophages) enable these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta), may be performed. Useful effector cells for suchassays include PBMC and NK cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., inan animal model such as that disclosed in Clynes et al., PNAS (USA)95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising antibody” refers to an antibody thatcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the antibody or by recombinant engineering of thenucleic acid encoding the antibody. Accordingly, a compositioncomprising an antibody having an Fc region according to this inventioncan comprise an antibody with K447, with all K447 removed, or a mixtureof antibodies with and without the K447 residue.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative and exemplary embodimentsfor measuring binding affinity are described in the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% TWEEN-20™ inPBS. When the plates have dried, 150 μl/well of scintillant(MICROSCINT-20™; Packard) is added, and the plates are counted on aTOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations ofeach Fab that give less than or equal to 20% of maximal binding arechosen for use in competitive binding assays.

According to another embodiment, the Kd or Kd value is measured by usingsurface plasmon resonance assays using a BIACORE®-2000 or aBIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CMS chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CMS, BIACORE, Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% TWEEN-20™ surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 106 M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on)”according to this invention can also be determined as described aboveusing a BIACORE®-2000 or a BIACORE®-3000 system (BIAcore, Inc.,Piscataway, N.J.).

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally, one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework or a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor a human consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. Where pre-existing amino acid changes arepresent in a VH, preferably those changes occur at only three, two, orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may be 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., supra. In one embodiment, for the VL, the subgroup issubgroup kappa I as in Kabat et al., supra. In one embodiment, for theVH, the subgroup is subgroup III as in Kabat et al., supra.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences:

(SEQ ID NO: 31) EVQLVESGGGLVQPGGSLRLSCAAS H1 (SEQ ID NO: 32)WVRQAPGKGLEWV H2 (SEQ ID NO: 33) RFTISADTSKNTLYLQMNSLRAEDTAVYYC H3(SEQ ID NO: 34) WGQGTLVTVSS.  See Figure 4.

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 35) DIQMTQSPSSLSASVGDRVTITC L1 (SEQ ID NO: 36)WYQQKPGKAPKLLIY  L2 (SEQ ID NO: 37) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC L3(SEQ ID NO: 38) FGQGTKVEIK.  See Figure 5.

As used herein, “codon set” refers to a set of different nucleotidetriplet sequences used to encode desired variant amino acids. A set ofoligonucleotides can be synthesized, for example, by solid phasesynthesis, including sequences that represent all possible combinationsof nucleotide triplets provided by the codon set and that will encodethe desired group of amino acids. A standard form of codon designationis that of the IUB code, which is known in the art and described herein.A codon set typically is represented by 3 capital letters in italics,e.g., NNK, NNS, XYZ, DVK and the like. A “non-random codon set,” as usedherein, thus refers to a codon set that encodes select amino acids thatfulfill partially, preferably completely, the criteria for amino acidselection as described herein. Synthesis of oligonucleotides withselected nucleotide “degeneracy” at certain positions is well known inthat art, for example, the TRIM approach (Knappek et al. (1999) J. Mol.Biol. 296:57-86); Garrard & Henner (1993) Gene 128:103). Such sets ofoligonucleotides having certain codon sets can be synthesized usingcommercial nucleic acid synthesizers (available from, for example,Applied Biosystems, Foster City, Calif.), or can be obtainedcommercially (for example, from Life Technologies, Rockville, Md.).Therefore, a set of oligonucleotides synthesized having a particularcodon set will typically include a plurality of oligonucleotides withdifferent sequences, the differences established by the codon set withinthe overall sequence. Oligonucleotides, as used according to theinvention, have sequences that allow for hybridization to a variabledomain nucleic acid template and also can, but does not necessarily,include restriction enzyme sites useful for, for example, cloningpurposes.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (for example, polypeptides of the invention), or thenucleic acids that encode these sequences, the sequences being differentin the combination of variant amino acids that are introduced into thesesequences according to the methods of the invention.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target antigen with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman (1992) Curr. Opin.Struct. Biol. 3:355-362, and references cited therein. In a monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells (1991) Methods: A companion to Methodsin Enzymology 3:205-0216.

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids, which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or aderivative thereof, or a lambdoid phage, such as lambda, 21, phi80,phi81, 82, 424, 434, etc., or a derivative thereof.

As used herein, “solvent accessible position” refers to a position of anamino acid residue in the variable regions of the heavy and light chainsof a source antibody or antigen binding fragment that is determined,based on structure, ensemble of structures and/or modeled structure ofthe antibody or antigen binding fragment, as potentially available forsolvent access and/or contact with a molecule, such as anantibody-specific antigen. These positions are typically found in theCDRs and on the exterior of the protein. The solvent accessiblepositions of an antibody or antigen binding fragment, as defined herein,can be determined using any of a number of algorithms known in the art.In one embodiment, solvent accessible positions are determined usingcoordinates from a 3-dimensional model of an antibody, preferably usinga computer program such as the InsightII program (Accelrys, San Diego,Calif.). Solvent accessible positions can also be determined usingalgorithms known in the art (e.g., Lee and Richards (1971) J. Mol. Biol.55, 379 and Connolly (1983) J. Appl. Cryst. 16, 548). Determination ofsolvent accessible positions can be performed using software suitablefor protein modeling and 3-dimensional structural information obtainedfrom an antibody. Software that can be utilized for these purposesincludes SYBYL Biopolymer Module software (Tripos Associates).Generally, where an algorithm (program) requires a user input sizeparameter, the “size” of a probe which is used in the calculation is setat about 1.4 Angstrom or smaller in radius. In addition, determinationof solvent accessible regions and area methods using software forpersonal computers has been described by Pacios (1994) Comput. Chem.18(4): 377-386.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promote angiogenesis, endothelialcell growth, stability of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, PlGF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, Delta-likeligand 4 (DLL4), Del-1, fibroblast growth factors: acidic (aFGF) andbasic (bFGF), Follistatin, Granulocyte colony-stimulating factor(G-CSF), Hepatocyte growth factor (HGF)/scatter factor (SF),Interleukin-8 (IL-8), Leptin, Midkine, neuropilins, Placental growthfactor, Platelet-derived endothelial cell growth factor (PD-ECGF),Platelet-derived growth factor, especially PDGF-BB or PDGFR-beta,Pleiotrophin (PTN), Progranulin, Proliferin, Transforming growthfactor-alpha (TGF-alpha), Transforming growth factor-beta (TGF-beta),Tumor necrosis factor-alpha (TNF-alpha), etc. It would also includefactors that accelerate wound healing, such as growth hormone,insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor(EGF), CTGF and members of its family, and TGF-alpha and TGF-beta. See,e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streitand Detmar (2003) Oncogene 22:3172-3179; Ferrara & Alitalo (1999) NatureMedicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556(e.g., Table 1 listing known angiogenic factors); and, Sato (2003) Int.J. Clin. Oncol. 8:200-206.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide (including, e.g., aninhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, arecombinant protein, an antibody, or conjugates or fusion proteinsthereof, that inhibits angiogenesis, vasculogenesis, or undesirablevascular permeability, either directly or indirectly. It should beunderstood that the anti-angiogenesis agent includes those agents thatbind and block the angiogenic activity of the angiogenic factor or itsreceptor. For example, an anti-angiogenesis agent is an antibody orother antagonist to an angiogenic agent as defined above, e.g.,antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptor orFlt-1 receptor), anti-PDGFR inhibitors such as Gleevec™ (ImatinibMesylate), small molecules that block VEGF receptor signaling (e.g.,PTK787/ZK2284, SU6668, SUTENT®/SU11248 (sunitinib malate), AMG706, orthose described in, e.g., international patent application WO2004/113304). Anti-angiogensis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003)Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapy inmalignant melanoma); Ferrara & Alitalo (1999) Nature Medicine5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table2 listing known antiangiogenic factors); and, Sato (2003) Int. J. Clin.Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used inclinical trials).

The term “VEGF” or “VEGF-A” as used herein refers to the 165-amino acidhuman vascular endothelial cell growth factor and related 121-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by Leung et al. (1989) Science 246:1306, and Houck et al.(1991) Mol. Endocrin, 5:1806, together with the naturally occurringallelic and processed forms thereof. The term “VEGF” also refers toVEGFs from non-human species such as mouse, rat, or primate. Sometimesthe VEGF from a specific species are indicated by terms such as hVEGFfor human VEGF, mVEGF for murine VEGF, etc. The term “VEGF” is also usedto refer to truncated forms of the polypeptide comprising amino acids 8to 109 or 1 to 109 of the 165-amino acid human vascular endothelial cellgrowth factor. Reference to any such forms of VEGF may be identified inthe present application, e.g., by “VEGF (8-109),” “VEGF (1-109)” or“VEGF₁₆₅.” The amino acid positions for a “truncated” native VEGF arenumbered as indicated in the native VEGF sequence. For example, aminoacid position 17 (methionine) in truncated native VEGF is also position17 (methionine) in native VEGF. The truncated native VEGF has bindingaffinity for the KDR and Flt-1 receptors comparable to native VEGF.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. In one embodiment, the anti-VEGFantibody of the invention is antibody B20-4.1.1. In yet anotherembodiment, the the anti-VEGF antibody of the invention is antibodyB20-4.1.1RR. In yet another embodiment, the anti-VEGF antibody of theinvention can be used as a therapeutic agent in targeting andinterfering with diseases or conditions wherein the VEGF activity isinvolved. An anti-VEGF antibody will usually not bind to other VEGFhomologues such as VEGF-B or VEGF-C, nor other growth factors such asPlGF, PDGF or bFGF.

The anti-VEGF antibody “Bevacizumab (BV),” also known as “rhuMAb VEGF”or “AVASTIN®,” is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al. (1997) Cancer Res. 57:4593-4599. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of Bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 daltons and isglycosylated.

The term “B20 series polypeptide” as used herein refers to apolypeptide, including an antibody that binds to VEGF. B20 seriespolypeptides include, but are not limited to, antibodies derived from asequence of the B20 antibody or a B20-derived antibody described in U.S.Publication No. 20060280747, U.S. Publication No. 20070141065 and/orU.S. Publication No. 20070020267, the content of these patentapplications are expressly incorporated herein by reference. In oneembodiment, B20 series polypeptide is B20-4.1 as described in U.S.Publication No. 20060280747, U.S. Publication No. 20070141065 and/orU.S. Publication No. 20070020267.

The term “B20-4.1.1” as used herein refers to that an antibody thatbinds to VEGF and comprises an antibody wherein HVR-H1 is SEQ ID NO:1;HVR-H2 is SEQ ID NO:2; HVR-H3 is SEQ ID NO:3; HVR-L1 is SEQ ID NO:4;HVR-L2 is SEQ ID NO:6; HVR-L3 is SEQ ID NO:7.

The term “B20-4.1.1RR” as used herein refers to that an antibody thatbinds to VEGF and comprises an antibody wherein HVR-H1 is SEQ ID NO:1;HVR-H2 is SEQ ID NO:2; HVR-H3 is SEQ ID NO:3; HVR-L1 is SEQ ID NO:5;HVR-L2 is SEQ ID NO:6; HVR-L3 is SEQ ID NO:7.

The term “G6-31” as used herein is one of the G-6 series polypeptides asdescribed in U.S. Publication No. 20060280747, U.S. Publication No.20070141065 and/or U.S. Publication No. 20070020267. Other G-6 seriespolypeptides, as described in U.S. Publication No. 20060280747, U.S.Publication No. 20070141065 and/or U.S. Publication No. 20070020267include, but not limited to, G6-8 and G6-23.

The term “biological activity” and “biologically active,” with regard toa VEGF polypeptide, refer to physical/chemical properties and biologicalfunctions associated with VEGF.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including, but not limited to, its binding to one or moreVEGF receptors. VEGF antagonists include, without limitation, anti-VEGFantibodies and antigen-binding fragments thereof, receptor molecules andderivatives which bind specifically to VEGF thereby sequestering itsbinding to one or more receptors, anti-VEGF receptor antibodies and VEGFreceptor antagonists such as small molecule inhibitors of the VEGFRtyrosine kinases. The term “VEGF antagonist,” as used herein,specifically includes molecules, including antibodies, antibodyfragments, other binding polypeptides, peptides, and non-peptide smallmolecules, that bind to VEGF and are capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with VEGF activities.Thus, the term “VEGF activities” specifically includes VEGF mediatedbiological activities (as hereinabove defined) of VEGF. In oneembodiment, the VEGF-antagonist is anti-VEGF antibody B20-4.1.1. In yetanother embodiment, the VEGF-antagonist is anti-VEGF antibodyB20-4.1.1RR.

As used herein, “treatment” (and variations such as “treat” or“treating”) refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder or to slow the progression of adisease or disorder.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

A “disorder” is any condition that would benefit from treatment (forexample, mammals who suffer from or need prophylaxis against abnormalangiogenesis (excessive, inappropriate or uncontrolled angiogenesis) orvascular permeability). This includes chronic and acute disorders ordiseases including those pathological conditions, which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include malignant and benign tumors; non-leukemiasand lymphoid malignancies; and, in particular, tumor (e.g., cancer)metastasis.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include, but are not limited to,squamous cell cancer, lung cancer (including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung), cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer (including gastrointestinal cancer andgastrointestinal stromal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, melanoma,superficial spreading melanoma, lentigo maligna melanoma, acrallentiginous melanomas, nodular melanomas, as well as B-cell lymphoma(including low grade/follicular non-Hodgkin's lymphoma (NHL); smalllymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediategrade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

An “individual,” “subject,” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, farm animals (such as cows), sport animals, pets (such ascats, dogs, and horses), primates, mice and rats. In certainembodiments, a mammal is a human.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations may be sterile.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount encompasses an amount in which anytoxic or detrimental effects of the substance/molecule are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,but not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount would be less than the therapeutically effective amount.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a VEGF polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent.” Examples of therapeutic agents (anti-cancer agents)include, but are limited to, e.g., chemotherapeutic agents, growthinhibitory agents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, andother-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g.,a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib(Tarceva™), platelet derived growth factor inhibitors (e.g., Gleevec™(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons,cytokines, antagonists (e.g., neutralizing antibodies) that bind to oneor more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS,APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive andorganic chemical agents, etc. Combinations thereof are also included inthe invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosed below.Other cytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “toxin” is any substance capable of having a detrimental effect on thegrowth or proliferation of a cell.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); combretastatin; folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R) (e.g., erlotinib (Tarceva™)); and VEGF-A that reduce cellproliferation; vaccines such as THERATOPE® vaccine and gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH(e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib,SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib oretoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®);CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosinekinase inhibitors; serine-threonine kinase inhibitors such as rapamycin(sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such aslonafarnib (SCH 6636, SARASAR); and pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON. toremifene; aromatase inhibitorsthat inhibit the enzyme aromatase, which regulates estrogen productionin the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane,formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, andARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides,particularly those which inhibit expression of genes in signalingpathways implicated in abherant cell proliferation, such as, forexample, PKC-alpha, Raf and H-Ras; ribozymes such as a VEGF expressioninhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor;vaccines such as gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2;LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; Vinorelbine andEsperamicins (see U.S. Pat. No. 4,675,187), and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingVEGF) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one which significantly reduces the percentage of cells (such as acell expressing VEGF) in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in Mendelsohn and Israel, eds., TheMolecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W.B. Saunders,Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel)are anticancer drugs both derived from the yew tree. Docetaxel(TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is asemisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb).Paclitaxel and docetaxel promote the assembly of microtubules fromtubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

The “pathology” of a disease includes all phenomena that compromise thewell-being of the patient. For cancer, this includes, withoutlimitation, abnormal or uncontrollable cell growth, metastasis,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels, suppressionor aggravation of inflammatory or immunological response, etc.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman (1986) “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfastand Stella et al. (1985). “Prodrugs: A Chemical Approach to TargetedDrug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press. The prodrugs of this invention include, but arenot limited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

“Purified” means that a molecule is present in a sample at aconcentration of at least 95% by weight, or at least 98% by weight ofthe sample in which it is contained.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isseparated from at least one other nucleic acid molecule with which it isordinarily associated, for example, in its natural environment. Anisolated nucleic acid molecule further includes a nucleic acid moleculecontained in cells that ordinarily express the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle-stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Y,where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “concurrently” is used herein to refer to administration of twoor more therapeutic agents, where at least part of the administrationoverlaps in time. Accordingly, concurrent administration includes adosing regimen when the administration of one or more agent(s) continuesafter discontinuing the administration of one or more other agent(s).

“Cancer recurrence” herein refers to a return of cancer followingtreatment. In one embodiment, cancer recurrence includes return ofcancer in the breast, as well as distant recurrence, where the cancerreturns outside of the breast.

Compositions of the Invention

The invention encompasses isolated antibody and polynucleotideembodiments. In one embodiment, an anti-VEGF antibody is purified.

This invention also encompasses compositions, including pharmaceuticalcompositions, comprising an anti-VEGF antibody; and polynucleotidescomprising sequences encoding an anti-VEGF antibody. As used herein,compositions comprise one or more antibodies that bind to VEGF, and/orone or more polynucleotides comprising sequences encoding one or moreantibodies that bind to VEGF. These compositions may further comprisesuitable carriers, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art.

In one embodiment, the anti-VEGF antibodies of the invention aremonoclonal. In yet another embodiment, the anti-VEGF antibodies arepolyclonal. Also encompassed within the scope of the invention are Fab,Fab′, Fab′-SH and F(ab′)₂ fragments of the anti-VEGF antibodies providedherein. These antibody fragments can be created by traditional means,such as enzymatic digestion, or may be generated by recombinanttechniques. Such antibody fragments may be chimeric or humanized. Thesefragments are useful for the diagnostic and purposes set forth below. Inone embodiment, an anti-VEGF antibody is a chimeric, humanized, or humanantibody.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

Exemplary monoclonal antibodies derived from a phage library areprovided herein and described in Example 1. Those antibodies aredesignated B20-4.1.1 and B20-4.1.1RR. The sequences of the heavy andlight chain variable domains of B20-4.1.1 and B20-4.1.1RR are shown inFIGS. 1 and 6-9.

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g., as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest. Further exemplaryembodiments of anti-VEGF antibodies are provided below.

Specific Embodiments of Anti-VEGF Antibodies

In one embodiment, the invention provides an antibody comprising:

(i) a HVR-H1 sequence comprising the sequence of SEQ ID NO:1;

(ii) a HVR-H2 sequence comprising the sequence of SEQ ID NO:2;

(iii) a HVR-H3 sequence comprising the sequence of SEQ ID NO:3.

(iv) a HVR-L1 sequence comprising the sequence of SEQ ID NO:4.

(v) a HVR-L2 sequence comprising the sequence of SEQ ID NO:6.

(vi) a HVR-L3 sequence comprising the sequence of SEQ ID NO:7.

In another embodiment, the invention provides an antibody comprising:

(i) a HVR-H1 sequence comprising the sequence of SEQ ID NO:1;

(ii) a HVR-H2 sequence comprising the sequence of SEQ ID NO:2;

(iii) a HVR-H3 sequence comprising the sequence of SEQ ID NO:3.

(iv) a HVR-L1 sequence comprising the sequence of SEQ ID NO:5.

(v) a HVR-L2 sequence comprising the sequence of SEQ ID NO:6.

(vi) a HVR-L3 sequence comprising the sequence of SEQ ID NO:7.

The amino acid sequences of SEQ ID NOs: 1 to 7 are numbered with respectto individual HVR (i.e., H1, H2 or H3) as indicated in FIG. 1, thenumbering being consistent with the Kabat numbering system as describedherein.

In one aspect, the invention provides antibodies comprising heavy chainHVR sequences as depicted in FIG. 1.

Some embodiments of antibodies of the invention comprise a light chainvariable domain of humanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN®,Genentech, Inc., South San Francisco, Calif., USA) (also referred to inU.S. Pat. No. 6,407,213 and Lee et al., J. Mol. Biol. (2004),340(5):1073-93) as depicted in SEQ ID NO:39 below.

(SEQ ID NO: 39) ^(l)Asp Ile Gln Met Thr Gln Ser Pro Ser Ser LeuSer Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Be Ser Ser Leu Gln Pro Glu Asp PheAla Thr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys¹⁰⁷(HVR residues are underlined).

In one embodiment, the huMAb4D5-8 light chain variable domain sequenceis modified at one or more of positions 30, 66 and 91 (Asn, Arg and Hisas indicated in bold/italics above, respectively). In one embodiment,the modified huMAb4D5-8 sequence comprises Ser in position 30, Gly inposition 66 and/or Ser in position 91. Accordingly, in one embodiment,an antibody of the invention comprises a light chain variable domaincomprising the sequence depicted in SEQ ID NO:40 below:

(SEQ ID NO: 40) ¹Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleTyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys¹⁰⁷(HVR residues are underlined).

Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics above.

Antibodies of the invention can comprise any suitable framework variabledomain sequence, provided binding activity to VEGF is substantiallyretained. For example, in some embodiments, antibodies of the inventioncomprise a human subgroup III heavy chain framework consensus sequence.In one embodiment of these antibodies, the framework consensus sequencecomprises substitution at position 71, 73 and/or 78. In some embodimentsof these antibodies, position 71 is A, 73 is T and/or 78 is A. In oneembodiment, these antibodies comprise heavy chain variable domainframework sequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc., SouthSan Francisco, Calif., USA) (also referred to in U.S. Pat. Nos.6,407,213 & 5,821,337, and Lee et al., J. Mol. Biol. (2004),340(5):1073-93). In one embodiment, these antibodies further comprise ahuman κI light chain framework consensus sequence. In one embodiment,these antibodies comprise light chain HVR sequences of huMAb4D5-8 asdescribed in U.S. Pat. Nos. 6,407,213 & 5,821,337). In one embodiment,these antibodies comprise light chain variable domain sequences ofhuMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South San Francisco, Calif.,USA) (also referred to in U.S. Pat. Nos. 6,407,213 & 5,821,337, and Leeet al., J. Mol. Biol. (2004), 340(5):1073-93).

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequencesof SEQ ID NOS:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, and/or 26 (FIGS. 2a and 2b ), and HVR H1, H2 and H3sequences are SEQ ID NOS:1, 2 and/or 3, respectively. In one embodiment,an antibody of the invention comprises a light chain variable domain,wherein the framework sequence comprises the sequences of SEQ ID NOS:27,28, 29 and/or 30 (FIG. 3), HVR L1 sequence is SEQ ID NOS:4 or 5, HVR L2sequence is SEQ ID NO:6, and HVR L3 sequence is SEQ ID NO:7.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:43. In oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NO:44 or 45. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:43 and a lightchain variable domain comprising the sequence of SEQ ID NO:45. In yetanother embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:44 and a lightchain variable domain comprising the sequence of SEQ ID NO:45.

In one aspect, the invention provides an antibody that competes with anyof the above-mentioned antibodies for binding to VEGF. In one aspect,the invention provides an antibody that binds to the same epitope onVEGF as any of the above-mentioned antibodies.

Antibody Fragments

The present invention encompasses antibody fragments. Antibody fragmentsmay be generated by traditional means, such as enzymatic digestion, orby recombinant techniques. In certain circumstances there are advantagesof using antibody fragments, rather than whole antibodies. The smallersize of the fragments allows for rapid clearance, and may lead toimproved access to solid tumors. For a review of certain antibodyfragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody. See, e.g., Sims et al.(1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol.196:901. Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. See, e.g., Carter et al. (1992) Proc.Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol.,151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human Antibodies

Human antibodies of the invention can be constructed by combining Fvclone variable domain sequence(s) selected from human-derived phagedisplay libraries with known human constant domain sequences(s) asdescribed above. Alternatively, human monoclonal antibodies of theinvention can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor, J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci U.S.A, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting,”either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is for VEGF andthe other is for any other antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of VEGF. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress VEGF. These antibodies possess a VEGF-binding arm and an armwhich binds a cytotoxic agent, such as, e.g., saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g., tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g., alanine or threonine). Thisprovides a mechanism for increasing the yield of the heterodimer overother unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking method. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. U.S.A, 90:6444-6448 (1993) has provided an alternative mechanismfor making bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g., tetravalent antibodies),which can be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. In certain embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In certain embodiments, amultivalent antibody comprises (or consists of) three to about eightantigen binding sites. In one such embodiment, a multivalent antibodycomprises (or consists of) four antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (for example, twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (for example, four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polyeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (for0-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. (1997) TIBTECH 15:26-32. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

For example, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. Such variants may have improved ADCC function. See, e.g., U.S.Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which further improve ADCC, forexample, substitutions at positions 298, 333, and/or 334 of the Fcregion (Eu numbering of residues). Such substitutions may occur incombination with any of the variations described above.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for many applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the antibody are measured to ensurethat only the desired properties are maintained. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-92 (1991). Non-limiting examples of invitro assays to assess ADCC activity of a molecule of interest isdescribed in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I., et al.Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (seeBruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. To assess complement activation, a CDCassaymay be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, for example, Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

Other antibody variants having one or more amino acid substitutions areprovided. Sites of interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions.” More substantial changes, denominated“exemplary substitutions” are provided in Table 1, or as furtherdescribed below in reference to amino acid classes. Amino acidsubstitutions may be introduced into an antibody of interest and theproducts screened, e.g., for a desired activity, such as improvedantigen binding, decreased immunogenicity, improved ADCC or CDC, etc.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Modifications in the biological properties of an antibody may beaccomplished by selecting substitutions that affect (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Amino acids may be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)):

-   -   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe        (F), Trp (W), Met (M)    -   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr        (Y), Asn (N), Gln (Q)    -   (3) acidic: Asp (D), Glu (E)    -   (4) basic: Lys (K), Arg (R), His (H)    -   Alternatively, naturally occurring residues may be divided into        groups based on common side-chain properties:        -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;        -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;        -   (3) acidic: Asp, Glu;        -   (4) basic: His, Lys, Arg;        -   (5) residues that influence chain orientation: Gly, Pro;        -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (e.g., non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. An exemplary substitutional variant is an affinity maturedantibody, which may be conveniently generated using phage display-basedaffinity maturation techniques. Briefly, several hypervariable regionsites (e.g., 6-7 sites) are mutated to generate all possible amino acidsubstitutions at each site. The antibodies thus generated are displayedfrom filamentous phage particles as fusions to at least part of a phagecoat protein (e.g., the gene III product of M13) packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g., binding affinity). In order to identifycandidate hypervariable region sites for modification, scanningmutagenesis (e.g., alanine scanning) can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according totechniques known in the art, including those elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening using techniques known in the art, including those describedherein, and variants with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g., a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g., in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter, Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al., J. Immunol. 164: 4178-4184 (2000).

In another aspect, the invention provides antibodies comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

In yet another aspect, it may be desirable to create cysteine engineeredantibodies, e.g., “thioMAbs,” in which one or more residues of anantibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, as described further herein. Incertain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region.

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Immunoconjugates

The invention also provides immunoconjugates (interchangeably referredto as “antibody-drug conjugates,” or “ADCs”) comprising an antibodyconjugated to one or more cytotoxic agents, such as a chemotherapeuticagent, a drug, a growth inhibitory agent, a toxin (e.g., a proteintoxin, an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Immunoconjugates have been used for the local delivery of cytotoxicagents, i.e., drugs that kill or inhibit the growth or proliferation ofcells, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion inPharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology23(9):1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos(1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer(1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278).Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and intracellular accumulation therein, where systemicadministration of unconjugated drugs may result in unacceptable levelsof toxicity to normal cells as well as the tumor cells sought to beeliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe(1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological And ClinicalApplications (A. Pinchera et al., eds) pp. 475-506. Both polyclonalantibodies and monoclonal antibodies have been reported as useful inthese strategies (Rowland et al., (1986) Cancer Immunol. Immunother.21:183-87). Drugs used in these methods include daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins usedin antibody-toxin conjugates include bacterial toxins such as diphtheriatoxin, plant toxins such as ricin, small molecule toxins such asgeldanamycin (Mandler et al (2000) J. Nat. Cancer Inst.92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791),maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928;Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins may exerttheir cytotoxic effects by mechanisms including tubulin binding, DNAbinding, or topoisomerase inhibition. Some cytotoxic drugs tend to beinactive or less active when conjugated to large antibodies or proteinreceptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and 111In or 90Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a huCD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5585089; 5606040;5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen,Inc.), an antibody-drug conjugate composed of the huC242 antibody linkedvia the disulfide linker SPP to the maytansinoid drug moiety, DM1, isadvancing into Phase II trials for the treatment of cancers that expressCanAg, such as colon, pancreatic, gastric, and other cancers. MLN-2704(Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drugconjugate composed of the anti-prostate specific membrane antigen (PSMA)monoclonal antibody linked to the maytansinoid drug moiety, DM1, isunder development for the potential treatment of prostate tumors. Theauristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE),synthetic analogs of dolastatin, were conjugated to chimeric monoclonalantibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specificto CD30 on hematological malignancies) (Doronina et al (2003) NatureBiotechnol. 21(7):778-784) and are under therapeutic development.

In certain embodiments, an immunoconjugate comprises an antibody and achemotherapeutic agent or other toxin. Chemotherapeutic agents useful inthe generation of immunoconjugates are described herein (e.g., above).Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹I, ⁹⁰Y, and¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×105 HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides,”volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc.111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277;Pettit, G. R., et al. Synthesis, 1996, 719-725; and Pettit et al. (1996)J. Chem. Soc. Perkin Trans. 1 5:859-863. See also Doronina (2003) NatBiotechnol 21(7):778-784; “Monomethylvaline Compounds Capable ofConjugation to Ligands,” U.S. Ser. No. 10/983,340, filed Nov. 5, 2004,hereby incorporated by reference in its entirety (disclosing, e.g.,linkers and methods of preparing monomethylvaline compounds such as MMAEand MMAF conjugated to linkers).

Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics is capable of producing double-stranded DNA breaksat sub-picomolar concentrations. For the preparation of conjugates ofthe calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all toAmerican Cyanamid Company). Structural analogues of calicheamicin whichmay be used include, but are not limited to, γ1I, α2I, α3I,N-acetyl-γ1I, PSAG and θI1 (Hinman et al., Cancer Research 53:3336-3342(1993), Lode et al., Cancer Research 58:2925-2928 (1998) and theaforementioned U.S. patents to American Cyanamid). Another anti-tumordrug that the antibody can be conjugated is QFA which is an antifolate.Both calicheamicin and QFA have intracellular sites of action and do notreadily cross the plasma membrane. Therefore, cellular uptake of theseagents through antibody mediated internalization greatly enhances theircytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc99m or 1123, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc⁹⁹m or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al. (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds expressly contemplate, but are not limited to, ADCprepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). Seepages 467-498, 2003-2004 Applications Handbook and Catalog.

Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC), an antibody (Ab) is conjugated toone or more drug moieties (D), e.g., about 1 to about 20 drug moietiesper antibody, through a linker (L). The ADC of Formula I may be preparedby several routes, employing organic chemistry reactions, conditions,and reagents known to those skilled in the art, including: (1) reactionof a nucleophilic group of an antibody with a bivalent linker reagent,to form Ab-L, via a covalent bond, followed by reaction with a drugmoiety D; and (2) reaction of a nucleophilic group of a drug moiety witha bivalent linker reagent, to form D-L, via a covalent bond, followed byreaction with the nucleophilic group of an antibody. Additional methodsfor preparing ADC are described herein.Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl(4-iodo-acetyl)aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzyme, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g., lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g.,by borohydride reagents to form stable amine linkages. In oneembodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehydes can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Certain Methods of Making Antibodies

Certain Hybridoma-Based Methods

Monoclonal antibodies of the invention can be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), andfurther described, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260(1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) regardinghuman-human hybridomas. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 regarding production of monoclonalhuman natural IgM antibodies from hybridoma cell lines. Human hybridomatechnology (Trioma technology) is described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.An exemplary protocol for producing monoclonal antibodies using thehybridoma method is described as follows. In one embodiment, a mouse orother appropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization. Antibodiesare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of a polypeptide comprising VEGF or a fragment thereof,and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalosedicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton,Mont.). A polypeptide comprising VEGF or a fragment thereof may beprepared using methods well known in the art, such as recombinantmethods, some of which are further described herein. Serum fromimmunized animals is assayed for anti-VEGF antibodies, and boosterimmunizations are optionally administered. Lymphocytes from animalsproducing anti-VEGF antibodies are isolated. Alternatively, lymphocytesmay be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies that bind to VEGF. The bindingspecificity of monoclonal antibodies produced by hybridoma cells may bedetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay(ELISA). The binding affinity of the monoclonal antibody can bedetermined, for example, by Scatchard analysis. See, e.g., Munson etal., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, hybridomacells may be grown in vivo as ascites tumors in an animal. Monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography. One procedure for isolation of proteins fromhybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method includes using minimal salts, such as lyotropicsalts, in the binding process and preferably also using small amounts oforganic solvents in the elution process.

Certain Library Screening Methods

Antibodies of the invention can be made by using combinatorial librariesto screen for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are describedgenerally in Hoogenboom et al. in Methods in Molecular Biology 178:1-37(O'Brien et al., ed., Human Press, Totowa, N.J., 2001). For example, onemethod of generating antibodies of interest is through the use of aphage antibody library as described in Lee et al., J. Mol. Biol. (2004),340(5):1073-93.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the antibodies of the invention can beobtained by designing a suitable antigen screening procedure to selectfor the phage clone of interest followed by construction of a fulllength antibody clone using the Fv sequences from the phage clone ofinterest and suitable constant region (Fc) sequences described in Kabatet al., Sequences of Proteins of Immunological Interest, Fifth Edition,NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g., as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g., as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-VEGF clones is desired, the subject is immunized withVEGF to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-VEGF clones isobtained by generating an anti-VEGF antibody response in transgenic micecarrying a functional human immunoglobulin gene array (and lacking afunctional endogenous antibody production system) such that VEGFimmunization gives rise to B cells producing human antibodies againstVEGF. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-VEGF reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing VEGF-specific membrane bound antibody, e.g., by cellseparation using VEGF affinity chromatography or adsorption of cells tofluorochrome-labeled VEGF followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which VEGF isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (U.S.A), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (U.S.A), 86:5728-5732 (1989). To maximize complementarity, degeneracy can beincorporated in the primers as described in Orlandi et al. (1989) orSastry et al. (1989). In certain embodiments, library diversity ismaximized by using PCR primers targeted to each V-gene family in orderto amplify all available VH and VL arrangements present in the immunecell nucleic acid sample, e.g. as described in the method of Marks etal., J. Mol. Biol., 222: 581-597 (1991) or as described in the method ofOrum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning ofthe amplified DNA into expression vectors, rare restriction sites can beintroduced within the PCR primer as a tag at one end as described inOrlandi et al. (1989), or by further PCR amplification with a taggedprimer as described in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. U.S.A, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci.U.S.A, 88: 7978-7982 (1991), or assembled together by PCR and thencloned, e.g. as described in Clackson et al., Nature, 352: 624-628(1991). PCR assembly can also be used to join VH and VL DNAs with DNAencoding a flexible peptide spacer to form single chain Fv (scFv)repertoires. In yet another technique, “in cell PCR assembly” is used tocombine VH and VL genes within lymphocytes by PCR and then clonerepertoires of linked genes as described in Embleton et al., Nucl. AcidsRes., 20: 3831-3837 (1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci U.S.A, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g., using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, VEGF can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized VEGF underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g., as described in Barbas et al., Proc.Natl. Acad. Sci U.S.A, 88: 7978-7982 (1991), or by alkali, e.g., asdescribed in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or byVEGF antigen competition, e.g., in a procedure similar to the antigencompetition method of Clackson et al., Nature, 352: 624-628 (1991).Phages can be enriched 20-1,000-fold in a single round of selection.Moreover, the enriched phages can be grown in bacterial culture andsubjected to further rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for VEGF.However, random mutation of a selected antibody (e.g., as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting VEGF, rare high affinity phage could be competed out. To retainall higher affinity mutants, phages can be incubated with excessbiotinylated VEGF, but with the biotinylated VEGF at a concentration oflower molarity than the target molar affinity constant for VEGF. Thehigh affinity-binding phages can then be captured by streptavidin-coatedparamagnetic beads. Such “equilibrium capture” allows the antibodies tobe selected according to their affinities of binding, with sensitivitythat permits isolation of mutant clones with as little as two-foldhigher affinity from a great excess of phages with lower affinity.Conditions used in washing phages bound to a solid phase can also bemanipulated to discriminate on the basis of dissociation kinetics.

Anti-VEGF clones may be selected based on activity. In certainembodiments, the invention provides anti-VEGF antibodies that bind toliving cells that naturally express VEGF. In one embodiment, theinvention provides anti-VEGF antibodies that block the binding between aVEGF ligand and VEGF, but do not block the binding between a VEGF ligandand a second protein. Fv clones corresponding to such anti-VEGFantibodies can be selected by (1) isolating anti-VEGF clones from aphage library as described above, and optionally amplifying the isolatedpopulation of phage clones by growing up the population in a suitablebacterial host; (2) selecting VEGF and a second protein against whichblocking and non-blocking activity, respectively, is desired; (3)adsorbing the anti-VEGF phage clones to immobilized VEGF; (4) using anexcess of the second protein to elute any undesired clones thatrecognize VEGF-binding determinants which overlap or are shared with thebinding determinants of the second protein; and (5) eluting the cloneswhich remain adsorbed following step (4). Optionally, clones with thedesired blocking/non-blocking properties can be further enriched byrepeating the selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the invention is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-VEGF antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g., asin the method of Morrison et al., Proc. Natl. Acad. Sci. U.S.A, 81:6851-6855 (1984)). DNA encoding a hybridoma- or Fv clone-derivedantibody or fragment can be further modified by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. In this manner, “chimeric” or“hybrid” antibodies are prepared that have the binding specificity ofthe Fv clone or hybridoma clone-derived antibodies of the invention.

Vectors, Host Cells, and Recombinant Methods

Antibodies may also be produced using recombinant methods. Forrecombinant production of an anti-VEGF antibody, nucleic acid encodingthe antibody is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

Signal Sequence Component

An antibody of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process a native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the polyA tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., HumanaPress, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g, in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Lemnaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NSO andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 255-268.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

Pharmaceutical Formulations and Dosages

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages. Generally, alleviation ortreatment of a disease or disorder involves the lessening of one or moresymptoms or medical problems associated with the disease or disorder. Inthe case of cancer, the therapeutically effective amount of the drug canaccomplish one or a combination of the following: reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., to decrease to someextent and/or stop) cancer cell infiltration into peripheral organs;inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. In someembodiments, a composition of this invention can be used to prevent theonset or reoccurrence of the disease or disorder in a subject or mammal.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments.

In certain embodiments, depending on the type and severity of thedisease, about 1 μg/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody isan initial candidate dosage for administration to the patient, whether,for example, by one or more separate administrations, or by continuousinfusion. In another embodiment, about 1 μg/kg to 15 mg/kg (e.g. 0.1mg/kg-10 mg/kg) of antibody is an initial candidate dosage foradministration to the patient. A typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs.

One exemplary dosage of the antibody would be in the range from about0.05 mg/kg to about 15 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg, 7.5 mg/kg, 10 mg/kg or 15 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e.g. every day, every three days, everyweek or every two to three weeks (e.g., such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). In one embodiment, dose of about 10 mg/kg is administeredevery three days. An initial higher loading dose, followed by one ormore lower doses may be administered. In one embodiment, an exemplarydosing regimen comprises administering an initial loading dose of about4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of theantibody. However, other dosage regimens may be useful.

In certain embodiments, dosing regimens discussed herein are used incombination with a chemotherapy regimen as the first line therapy fortreating metastatic colorectal cancer. In some aspects, the chemotherapyregimen involves the traditional high-dose intermittent administration.In some other aspects, the chemotherapeutic agents are administeredusing smaller and more frequent doses without scheduled breaks(“metronomic chemotherapy”).

The progress of the therapy of the invention is easily monitored byconventional techniques and assays.

An antibody of the invention (and any additional therapeutic agent oradjuvant) can be administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Inaddition, the antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. Dosing can be by anysuitable route, e.g., by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

Pharmaceutical formulations herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

Pharmaceutical formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington: The Science and Practice of Pharmacy 20thedition (2000)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Methods

Therapeutic Methods

An antibody of the invention may be used in, for example, in vitro, exvivo, and in vivo therapeutic methods.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder (e.g., disorderassociated with increased expression and/or activity of VEGF) comprisingadministering an effective amount of an anti-VEGF antibody to a subjectin need of such treatment.

In one aspect, the invention provides methods for reducing, inhibiting,blocking, or preventing growth of a tumor or cancer, the methodscomprising administering an effective amount of an anti-VEGF antibody toa subject in need of such treatment.

In one aspect, the invention provides methods for inhibitingangiogenesis comprising administering an effective amount of ananti-VEGF antibody to a subject in need of such treatment.

In one aspect, the invention provides methods for inhibiting vascularpermeability comprising administering an effective amount of ananti-VEGF antibody to a subject in need of such treatment.

In one aspect, the invention provides methods for treating apathological condition associated with angiogenesis comprisingadministering an effective amount of an anti-VEGF antibody to a subjectin need of such treatment. In some embodiments, the pathologicalcondition associated with angiogenesis is a tumor, a cancer, and/or acell proliferative disorder.

An antibody of the invention can be administered to a human fortherapeutic purposes. In one embodiment, an antibody of the invention isused in a method for binding VEGF in an individual suffering from adisorder associated with increased VEGF expression and/or activity, themethod comprising administering to the individual the antibody such thatVEGF in the individual is bound. In one embodiment, the VEGF is humanVEGF, and the individual is a human individual. Alternatively, theindividual can be a mammal expressing VEGF to which an antibody of theinvention binds. Still further the individual can be a mammal into whichVEGF has been introduced (e.g., by administration of VEGF or byexpression of a transgene encoding VEGF).

In one aspect, at least some of the antibodies of the invention can bindVEGF from species other than human. Accordingly, the antibodies of theinvention can be used to bind specific antigen activity, e.g., in a cellculture containing the antigen, in human subjects or in other mammaliansubjects having the antigen with which an antibody of the inventioncross-reacts (e.g., chimpanzee, baboon, marmoset, cynomolgus and rhesus,pig or mouse). In one embodiment, the antibody of the invention can beused for inhibiting antigen activities by contacting the antibody withthe antigen such that antigen activity is inhibited. Preferably, theantigen is a human protein molecule.

Moreover, an antibody of the invention can be administered to anon-human mammal expressing VEGF with which the antibody cross-reacts(e.g., a primate, pig, rat, or mouse) for veterinary purposes or as ananimal model of human disease. Regarding the latter, such animal modelsmay be useful for evaluating the therapeutic efficacy of antibodies ofthe invention (e.g., testing of dosages and time courses ofadministration).

The antibodies of the invention can be used to treat, inhibit, delayprogression of, prevent/delay recurrence of, ameliorate, or preventdiseases, disorders or conditions associated with expression and/oractivity of one or more antigen molecules.

The present invention encompasses the prevention and treatment of tumormetastasis and anti-angiogenic cancer therapy, a novel cancer treatmentstrategy aimed at inhibiting the development of tumor blood vesselsrequired for providing nutrients to support tumor growth. The inventionspecifically includes inhibiting the neoplastic growth of tumor at theprimary site as well as preventing and/or treating metastasis of tumorsat the secondary sites, therefore allowing attack of the tumors by othertherapeutics. Examples of cancer to be treated (including prevention)herein include, but are not limited to, carcinoma, lymphoma, blastoma,sarcoma, and leukemia or lymphoid malignancies. More particular examplesof such cancers include squamous cell cancer (e.g., epithelial squamouscell cancer), lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer and gastrointestinalstromal cancer, pancreatic cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, cancer of the urinarytract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma,superficial spreading melanoma, lentigo maligna melanoma, acrallentiginous melanomas, nodular melanomas, multiple myeloma and B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), Meigs' syndrome,brain, as well as head and neck cancer, and associated metastases. Incertain embodiments, cancers that are amenable to treatment by theantibodies of the invention include breast cancer, colorectal cancer,rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL),renal cell cancer, prostate cancer, liver cancer, pancreatic cancer,soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head andneck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In someembodiments, the cancer is selected from the group consisting of smallcell lung cancer, neuroblastomas, melanoma, breast carcinoma, gastriccancer, colorectal cancer (CRC), and hepatocellular carcinoma. Yet, insome embodiments, the cancer is selected from the group consisting ofnon-small cell lung cancer, colorectal cancer and breast carcinoma,including metastatic forms of those cancers.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with one or more cytotoxic agent(s) is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thetarget cell to which it binds. In one embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the target cell. In oneembodiment, the cytotoxic agent targets or interferes with microtubulepolymerization. Examples of such cytotoxic agents include any of thechemotherapeutic agents noted herein (such as a maytansinoid,auristatin, dolastatin, or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

When the binding target of an antibody is located in the brain, certainembodiments of the invention provide for the antibody to traverse theblood-brain barrier. Several art-known approaches exist for transportingmolecules across the blood-brain barrier, including, but not limited to,physical methods, lipid-based methods, stem cell-based methods, andreceptor and channel-based methods.

Physical methods of transporting an antibody across the blood-brainbarrier include, but are not limited to, circumventing the blood-brainbarrier entirely, or by creating openings in the blood-brain barrier.Circumvention methods include, but are not limited to, direct injectioninto the brain (see, e.g., Papanastassiou et al., Gene Therapy 9:398-406 (2002)), interstitial infusion/convection-enhanced delivery(see, e.g., Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080(1994)), and implanting a delivery device in the brain (see, e.g., Gillet al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers™, GuildfordPharmaceutical). Methods of creating openings in the barrier include,but are not limited to, ultrasound (see, e.g., U.S. Patent PublicationNo. 2002/0038086), osmotic pressure (e.g., by administration ofhypertonic mannitol (Neuwelt, E. A., Implication of the Blood-BrainBarrier and its Manipulation, Vols 1 & 2, Plenum Press, N.Y. (1989)),permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g.,U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416), andtransfection of neurons that straddle the blood-brain barrier withvectors containing genes encoding the antibody (see, e.g., U.S. PatentPublication No. 2003/0083299).

Lipid-based methods of transporting an antibody across the blood-brainbarrier include, but are not limited to, encapsulating the antibody inliposomes that are coupled to antibody binding fragments that bind toreceptors on the vascular endothelium of the blood-brain barrier (see,e.g., U.S. Patent Application Publication No. 20020025313), and coatingthe antibody in low-density lipoprotein particles (see, e.g., U.S.Patent Application Publication No. 20040204354) or apolipoprotein E(see, e.g., U.S. Patent Application Publication No. 20040131692).

Stem-cell based methods of transporting an antibody across theblood-brain barrier entail genetically engineering neural progenitorcells (NPCs) to express the antibody of interest and then implanting thestem cells into the brain of the individual to be treated. See Behrstocket al. (2005) Gene Ther. 15 Dec. 2005 advanced online publication(reporting that NPCs genetically engineered to express the neurotrophicfactor GDNF reduced symptoms of Parkinson disease when implanted intothe brains of rodent and primate models).

Receptor and channel-based methods of transporting an antibody acrossthe blood-brain barrier include, but are not limited to, usingglucocorticoid blockers to increase permeability of the blood-brainbarrier (see, e.g., U.S. Patent Application Publication Nos.2002/0065259, 2003/0162695, and 2005/0124533); activating potassiumchannels (see, e.g., U.S. Patent Application Publication No.2005/0089473), inhibiting ABC drug transporters (see, e.g., U.S. PatentApplication Publication No. 2003/0073713); coating antibodies with atransferrin and modulating activity of the one or more transferrinreceptors (see, e.g., U.S. Patent Application Publication No.2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat. No.5,004,697).

It is understood that any of the above therapeutic methods may becarried out using an immunoconjugate of the invention in place of or inaddition to an anti-VEGF antibody.

Combination Therapies

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with another antibody, chemotherapeuticagent(s) (including cocktails of chemotherapeutic agents), othercytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growthinhibitory agent(s). Where an antibody of the invention inhibits tumorgrowth, it may be particularly desirable to combine it with one or moreother therapeutic agent(s) which also inhibits tumor growth, e.g.,anti-angiogenic agents and/or chemotherapeutic agents. Alternatively, oradditionally, the patient may receive combined radiation therapy (e.g.external beam irradiation or therapy with a radioactive labeled agent,such as an antibody). Such combined therapies noted above includecombined administration (where the two or more agents are included inthe same or separate formulations), and separate administration, inwhich case, administration of the antibody of the invention can occurprior to, and/or following, administration of the adjunct therapy ortherapies.

In one embodiment, an antibody of the invention may be co-administeredwith at least one additional therapeutic agent and/or adjuvant. Forexample, anti-VEGF antibodies are used in combinations with anti-cancertherapeutics or anti-neovascularization therapeutics to treat variousneoplastic or non-neoplastic conditions. In one embodiment, theneoplastic or non-neoplastic condition is characterized by pathologicaldisorder associated with aberrant or undesired angiogenesis. Theanti-VEGF antibody can be administered serially or in combination withanother agent that is effective for those purposes, either in the samecomposition or as separate compositions. Alternatively, or additionally,multiple inhibitors of VEGF can be administered.

Typically, the anti-VEGF antibodies and anti-cancer agents are suitablefor the same or similar diseases to block or reduce a pathologicaldisorder such as a tumor, a cancer or a cell proliferative disorder. Inone embodiment the anti-cancer agent is an anti-angiogenic agent.

Many anti-angiogenic agents have been identified and are known in thearts, including those listed herein, e.g., listed under “Definitions,”and by, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara etal., Nature Reviews: Drug Discovery, 3:391-400 (2004); and Sato Int. J.Clin. Oncol., 8:200-206 (2003). See also, US Patent ApplicationUS20030055006.

In one embodiment, an anti-VEGF of the present invention is used incombination with one or more anti-VEGF neutralizing antibody (orfragment), antagonist to another VEGF family (e.g., VEGF-B, VEGF-C,VEGF-D, placental growth factor (PLGF)) or a VEGF receptor antagonistincluding, but not limited to, for example, soluble VEGF receptor (e.g.,VEGFR-1, VEGFR-2, VEGFR-3, neuropillins (e.g., NRP1, NRP2)) fragments,aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFRantibodies, low molecule weight inhibitors of VEGFR tyrosine kinases(RTK), antisense strategies for VEGF, ribozymes against VEGF or VEGFreceptors, antagonist variants of VEGF; and any combinations thereof.

In another embodiment, the anti-VEGF antibody of the invention can beused in combination with small molecule receptor tyrosine kinaseinhibitors (RTKIs) that target one or more tyrosine kinase receptorssuch as VEGF receptors, FGF receptors, EGF receptors and PDGF receptors.Many therapeutic small molecule RTKIs are known in the art, including,but are not limited to, vatalanib (PTK787), erlotinib (TARCEVA®),OSI-7904, ZD6474 (ZACTIMA®), ZD6126 (ANG453), ZD1839, sunitinib(SUTENT®), semaxanib (SU5416), AMG706, AG013736, Imatinib (GLEEVEC®),MLN-518, CEP-701, PKC-412, Lapatinib (GSK572016), VELCADE®, AZD2171,sorafenib (NEXAVAR®), XL880, and CHIR-265.

Other therapeutic agents useful for combination tumor therapy with theantibody of the invention include antagonist of other factors that areinvolved in tumor growth, such as EGFR, ErbB2 (also known as Her2)ErbB3, ErbB4, or TNF.

In certain embodiments, two or more angiogenesis inhibitors mayoptionally be co-administered to the patient in addition to VEGFantagonist and other agent. In yet another embodiment, one or moreadditional therapeutic agents, e.g., anti-cancer agents, can beadministered in combination with an anti-VEGF antibody of the presentinvention, an antagonist to another VEGF family, and/or ananti-angiogenic agent.

In certain aspects of the invention, other therapeutic agents useful forcombination tumor therapy with an anti-VEGF-antibody include othercancer therapies, (e.g., surgery, radiological treatments (e.g.,involving irradiation or administration of radioactive substances),chemotherapy, treatment with anti-cancer agents listed herein and knownin the art, or combinations thereof). An exemplary and non-limiting listof chemotherapeutic agents contemplated is provided herein under“Definitions.”

Alternatively, or additionally, two or more antibodies binding the sameor two or more different antigens disclosed herein can beco-administered to the patient. Sometimes, it may be beneficial to alsoadminister one or more cytokines to the patient.

The effective amounts of therapeutic agents administered in combinationwith an anti-VEGF antibody will be at the physician's or veterinarian'sdiscretion. Dosage administration and adjustment is done to achievemaximal management of the conditions to be treated. The dose willadditionally depend on such factors as the type of therapeutic agent tobe used and the specific patient being treated. Suitable dosages for theanti-cancer agent are those presently used and can be lowered due to thecombined action (synergy) of the anti-cancer agent and the anti-VEGFantibody. In certain embodiments, the combination of the inhibitorspotentiates the efficacy of a single inhibitor. The term “potentiate”refers to an improvement in the efficacy of a therapeutic agent at itscommon or approved dose. See also the section entitled Pharmaceuticalformulations and dosages herein.

Chemotherapeutic Agents

In one aspect, the invention provides a method of treating a disorder(such as a tumor, a cancer, or a cell proliferative disorder) byadministering effective amounts of an anti-VEGF antibody and/or anangiogenesis inhibitor(s) and one or more chemotherapeutic agents. Avariety of chemotherapeutic agents may be used in the combined treatmentmethods of the invention. An exemplary and non-limiting list ofchemotherapeutic agents contemplated is provided herein under“Definitions.” The administration of the anti-VEGF antibody and thechemotherapeutic agent can be done simultaneously, e.g., as a singlecomposition or as two or more distinct compositions, using the same ordifferent administration routes. Alternatively, or additionally, theadministration can be done sequentially, in any order. Alternatively, oradditionally, the steps can be performed as a combination of bothsequentially and simultaneously, in any order. In certain embodiments,intervals ranging from minutes to days, to weeks to months, can bepresent between the administrations of the two or more compositions. Forexample, the chemotherapeutic agent may be administered first, followedby the anti-VEGF antibody. However, simultaneous administration oradministration of the anti-VEGF antibody first is also contemplated.Accordingly, in one aspect, the invention provides methods comprisingadministration of an anti-VEGF antibody (such as B20-4.1.1 antibody orB20-4.1.1RR antibody), followed by administration of a chemotherapeuticagent. In certain embodiments, intervals ranging from minutes to days,to weeks to months, can be present between the administrations of thetwo or more compositions.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. Variation in dosage will likely occur depending onthe condition being treated. The physician administering treatment willbe able to determine the appropriate dose for the individual subject.

Relapse Tumor Growth

The invention also provides methods and compositions for inhibiting orpreventing relapse tumor growth or relapse cancer cell growth. Relapsetumor growth or relapse cancer cell growth is used to describe acondition in which patients undergoing or treated with one or morecurrently available therapies (e.g., cancer therapies, such aschemotherapy, radiation therapy, surgery, hormonal therapy and/orbiological therapy/immunotherapy, anti-VEGF antibody therapy,particularly a standard therapeutic regimen for the particular cancer)is not clinically adequate to treat the patients or the patients are nolonger receiving any beneficial effect from the therapy such that thesepatients need additional effective therapy. As used herein, the phrasecan also refer to a condition of the “non-responsive/refractory”patient, e.g., which describe patients who respond to therapy yet sufferfrom side effects, develop resistance, do not respond to the therapy, donot respond satisfactorily to the therapy, etc. In various embodiments,a cancer is relapse tumor growth or relapse cancer cell growth where thenumber of cancer cells has not been significantly reduced, or hasincreased, or tumor size has not been significantly reduced, or hasincreased, or fails any further reduction in size or in number of cancercells. The determination of whether the cancer cells are relapse tumorgrowth or relapse cancer cell growth can be made either in vivo or invitro by any method known in the art for assaying the effectiveness oftreatment on cancer cells, using the art-accepted meanings of “relapse”or “refractory” or “non-responsive” in such a context. A tumor resistantto anti-VEGF treatment is an example of a relapse tumor growth.

The invention provides methods of blocking or reducing relapse tumorgrowth or relapse cancer cell growth in a subject by administering oneor more anti-VEGF antibody of the present invention to block or reducethe relapse tumor growth or relapse cancer cell growth in subject. Incertain embodiments, the antibody can be administered subsequent to thecancer therapeutic. In certain embodiments, the anti-VEGF antibodies areadministered simultaneously with cancer therapy. Alternatively, oradditionally, the anti-VEGF antibody therapy alternates with anothercancer therapy, which can be performed in any order. The invention alsoencompasses methods for administering one or more inhibitory antibodiesto prevent the onset or recurrence of cancer in patients predisposed tohaving cancer. Generally, the subject was or is concurrently undergoingcancer therapy. In one embodiment, the cancer therapy is treatment withan anti-angiogenesis agent, e.g., a VEGF-C antagonist. Theanti-angiogenesis agent includes, but not limited to, those known in theart and those found under the “Definitions” herein. In one embodiment,the anti-angiogenesis agent is an anti-VEGF neutralizing antibody orfragment (e.g., humanized A4.6.1, AVASTIN® (Genentech, South SanFrancisco, Calif.), Y0317, M4, G6, B20, 2C3, etc.). See, e.g., U.S. Pat.Nos. 6,582,959, 6,884,879, 6,703,020; WO98/45332; WO 96/30046;WO94/10202; EP 0666868B1; US Patent Applications 20030206899,20030190317, 20030203409, and 20050112126; Popkov et al., Journal ofImmunological Methods 288:149-164 (2004); and, WO2005012359. Additionalagents can be administered in combination with anti-VEGF antibody forblocking or reducing relapse tumor growth or relapse cancer cell growth,e.g., see section entitled Combination Therapies herein.

Diagnostic Methods and Methods of Detection

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of VEGF. In certain embodiments,the method comprises contacting a test cell with an anti-VEGF antibody;determining the level of expression (either quantitatively orqualitatively) of VEGF by the test cell by detecting binding of theanti-VEGF antibody to VEGF; and comparing the level of expression ofVEGF by the test cell with the level of expression of VEGF by a controlcell (e.g., a normal cell of the same tissue origin as the test cell ora cell that expresses VEGF at levels comparable to such a normal cell),wherein a higher level of expression of VEGF by the test cell ascompared to the control cell indicates the presence of a disorderassociated with increased expression of VEGF. In certain embodiments,the test cell is obtained from an individual suspected of having adisorder associated with increased expression of VEGF. In certainembodiments, the disorder is a tumor, cancer, and/or cell proliferativedisorder.

Exemplary disorders that may be diagnosed using an antibody of theinvention include, but not limited to, squamous cell cancer, small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung,squamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric cancer, gastrointestinal cancer, gastrointestinalstromal cancer, pancreatic cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer,colon cancer, colorectal cancer, endometrial or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, liver cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma and varioustypes of head and neck cancer, melanoma, superficial spreading melanoma,lentigo maligna melanoma, acral lentiginous melanomas, nodularmelanomas, B-cell lymphoma, chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; post-transplant lymphoproliferative disorder (PTLD), abnormalvascular proliferation associated with phakomatoses, edema associatedwith brain tumors, and Meigs' syndrome.

In another aspect, the invention provides a complex of any of theanti-VEGF antibodies described herein and VEGF. In some embodiments, thecomplex is in vivo or in vitro. In some embodiments, the complexcomprises a cancer cell.

In another aspect, the invention provides a method of detecting thepresence of VEGF in a biological sample. The term “detecting” as usedherein encompasses quantitative or qualitative detection.

In certain embodiments, the method comprises contacting the biologicalsample with an anti-VEGF antibody under conditions permissive forbinding of the anti-VEGF antibody to VEGF, and detecting whether acomplex is formed between the anti-VEGF antibody and VEGF.

Anti-VEGF antibodies can be used for the detection of VEGF in any one ofa number of well known detection assay methods. For example, abiological sample may be assayed for VEGF by obtaining the sample from adesired source, admixing the sample with anti-VEGF antibody to allow theantibody to form antibody/VEGF complex with any VEGF present in themixture, and detecting any antibody/VEGF complex present in the mixture.The biological sample may be prepared for assay by methods known in theart which are suitable for the particular sample. The methods ofadmixing the sample with antibodies and the methods of detectingantibody/VEGF complex are chosen according to the type of assay used.

Analytical methods for VEGF all use one or more of the followingreagents: labeled VEGF analogue, immobilized VEGF analogue, labeledanti-VEGF antibody, immobilized anti-VEGF antibody and/or stericconjugates. The labeled reagents also are known as “tracers.”

In certain embodiments, the anti-VEGF antibody is detectably labeled.The label used is any detectable functionality that does not interferewith the binding of VEGF and anti-VEGF antibody. Labels include, but arenot limited to, labels or moieties that are detected directly (such asfluorescent, chromophoric, electron-dense, chemiluminescent, andradioactive labels), as well as moieties, such as enzymes or ligands,that are detected indirectly, e.g., through an enzymatic reaction ormolecular interaction. Exemplary labels include, but are not limited to,the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David etal., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol.Methods, 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem.,30: 407-412 (1982). Preferred labels herein are enzymes such ashorseradish peroxidase and alkaline phosphatase. The conjugation of suchlabel, including the enzymes, to the antibody is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., “Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y., 1981), pp. 147-166.

In certain embodiments, antibodies are immobilized on an insolublematrix. Immobilization may entail separating an anti-VEGF antibody fromany VEGF that remains free in solution. This conventionally isaccomplished by either insolubilizing the anti-VEGF antibody before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), or by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing theanti-VEGF antibody after formation of a complex between the anti-VEGFantibody and VEGF, e.g., by immunoprecipitation.

Assays used to detect binding of anti-VEGF antibodies to VEGF include,but not limited to, antigen-binding assays that are well known in theart, such as western blots, radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), competitive and “sandwich” assays,immunoprecipitation assays, fluorescent immunoassays, protein Aimmunoassays, immunohistochemistry (IHC) and steric inhibition assays.

In one embodiment, the expression of proteins in a sample may beexamined using immunohistochemistry and staining protocols.Immunohistochemical staining of tissue sections has been shown to be areliable method of assessing or detecting presence of proteins in asample. Immunohistochemistry (“IHC”) techniques utilize an antibody toprobe and visualize cellular antigens in situ, generally by chromogenicor fluorescent methods. For sample preparation, a tissue or cell samplefrom a mammal (typically a human patient) may be used. Examples ofsamples include, but are not limited to, cancer cells such as colon,breast, prostate, ovary, lung, stomach, pancreas, lymphoma, and leukemiacancer cells. The sample can be obtained by a variety of proceduresknown in the art including, but not limited to surgical excision,aspiration or biopsy. The tissue may be fresh or frozen. In oneembodiment, the sample is fixed and embedded in paraffin or the like.The tissue sample may be fixed (i.e., preserved) by conventionalmethodology. One of ordinary skill in the art will appreciate that thechoice of a fixative is determined by the purpose for which the sampleis to be histologically stained or otherwise analyzed. One of ordinaryskill in the art will also appreciate that the length of fixationdepends upon the size of the tissue sample and the fixative used.

IHC may be performed in combination with additional techniques such asmorphological staining and/or fluorescence in-situ hybridization. Twogeneral methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigen(e.g., VEGF) is determined directly. This direct assay uses a labeledreagent, such as a fluorescent tag or an enzyme-labeled primaryantibody, which can be visualized without further antibody interaction.In a typical indirect assay, unconjugated primary antibody binds to theantigen and then a labeled secondary antibody binds to the primaryantibody. Where the secondary antibody is conjugated to an enzymaticlabel, a chromogenic or fluorogenic substrate is added to providevisualization of the antigen. Signal amplification occurs becauseseveral secondary antibodies may react with different epitopes on theprimary antibody. The primary and/or secondary antibody used forimmunohistochemistry typically will be labeled with a detectable moiety.

Aside from the sample preparation procedures discussed above, furthertreatment of the tissue section prior to, during or following IHC may bedesired. For example, epitope retrieval methods, such as heating thetissue sample in citrate buffer may be carried out (see, e.g., Leong etal. Appl. Immunohistochem. 4(3):201 (1996)).

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the target proteinantigen in the tissue sample. Appropriate conditions for achieving thiscan be determined by routine experimentation. The extent of binding ofantibody to the sample is determined by using any one of the detectablelabels discussed above. Preferably, the label is an enzymatic label(e.g., HRPO) which catalyzes a chemical alteration of the chromogenicsubstrate such as 3,3′-diaminobenzidine chromogen. Preferably theenzymatic label is conjugated to antibody which binds specifically tothe primary antibody (e.g., the primary antibody is rabbit polyclonalantibody and secondary antibody is goat anti-rabbit antibody).

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g., using a microscope, and stainingintensity criteria, routinely used in the art, may be employed. Stainingintensity criteria may be evaluated as follows:

TABLE 2 Staining Pattern Score No staining is observed in cells. 0 Faint/barely perceptible staining is detected in more 1+ than 10% of thecells. Weak to moderate staining is observed in more than 2+ 10% of thecells. Moderate to strong staining is observed in more than 3+ 10% ofthe cells.

Typically, a staining pattern score of about 2+ or higher in an IHCassay is diagnostic and/or prognostic. In some embodiments, a stainingpattern score of about 1+ or higher is diagnostic and/or prognostic. Inother embodiments, a staining pattern score of about 3 of higher isdiagnostic and/or prognostic. It is understood that when cells and/ortissue from a tumor or colon adenoma are examined using IHC, staining isgenerally determined or assessed in tumor cell and/or tissue (as opposedto stromal or surrounding tissue that may be present in the sample).

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer VEGF analogue tocompete with the test sample VEGF for a limited number of anti-VEGFantibody antigen-binding sites. The anti-VEGF antibody generally isinsolubilized before or after the competition and then the tracer andVEGF bound to the anti-VEGF antibody are separated from the unboundtracer and VEGF. This separation is accomplished by decanting (where thebinding partner was preinsolubilized) or by centrifuging (where thebinding partner was precipitated after the competitive reaction). Theamount of test sample VEGF is inversely proportional to the amount ofbound tracer as measured by the amount of marker substance.Dose-response curves with known amounts of VEGF are prepared andcompared with the test results to quantitatively determine the amount ofVEGF present in the test sample. These assays are called ELISA systemswhen enzymes are used as the detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theVEGF is prepared and used such that when anti-VEGF antibody binds to theVEGF the presence of the anti-VEGF antibody modifies the enzymeactivity. In this case, the VEGF or its immunologically active fragmentsare conjugated with a bifunctional organic bridge to an enzyme such asperoxidase. Conjugates are selected for use with anti-VEGF antibody sothat binding of the anti-VEGF antibody inhibits or potentiates theenzyme activity of the label. This method per se is widely practicedunder the name of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small VEGF fragment so that antibody tohapten is substantially unable to bind the conjugate at the same time asanti-VEGF antibody. Under this assay procedure the VEGF present in thetest sample will bind anti-VEGF antibody, thereby allowing anti-haptento bind the conjugate, resulting in a change in the character of theconjugate hapten, e.g., a change in fluorescence when the hapten is afluorophore.

Sandwich assays particularly are useful for the determination of VEGF oranti-VEGF antibodies. In sequential sandwich assays an immobilizedanti-VEGF antibody is used to adsorb test sample VEGF, the test sampleis removed as by washing, the bound VEGF is used to adsorb a second,labeled anti-VEGF antibody and bound material is then separated fromresidual tracer. The amount of bound tracer is directly proportional totest sample VEGF. In “simultaneous” sandwich assays the test sample isnot separated before adding the labeled anti-VEGF. A sequential sandwichassay using an anti-VEGF monoclonal antibody as one antibody and apolyclonal anti-VEGF antibody as the other is useful in testing samplesfor VEGF.

The foregoing are merely exemplary detection assays for VEGF. Othermethods now or hereafter developed that use anti-VEGF antibody for thedetermination of VEGF are included within the scope hereof.

It is understood that any of the above embodiments of diagnosis ordetection may be carried out using an immunoconjugate of the inventionin place of or in addition to an anti-VEGF antibody.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or combined with another composition effective fortreating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody or immunoconjugate of the invention. The label or packageinsert indicates that the composition is used for treating the conditionof choice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody or immunoconjugate of the invention; and (b) asecond container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

The following examples are intended merely to illustrate the practice ofthe present invention and are not provided by way of limitation. Thedisclosures of all patent and scientific literatures cited herein areexpressly incorporated in their entirety by reference.

EXAMPLES Example 1 Generation and Characterization of Anti-VEGFAntibodies

Synthetic phage antibody libraries were built on a single framework(humanized anti-ErbB2 antibody, 4D5) by introducing diversity within thecomplementarity-determining regions (CDRs) of heavy and light chains(Lee, C. V. et al. J Mol Biol 340, 1073-93 (2004)). In brief,phage-displayed synthetic antibody libraries were built on a singlehuman framework by introducing synthetic diversity at solvent-exposedpositions within the heavy chain complementarity-determining regions(CDRs). To improve library performance, monovalent and bivalentantigen-binding fragment (Fab) libraries were constructed, and exploreddifferent CDR-H3 diversities by varying the amino acid composition andCDR length. The library was then expanded by increasing the variabilityof CDR-H3 length and using tailored codons that mimicked the amino acidcomposition of natural CDR-H3 sequences. Using these libraries withcompletely synthetic CDRs displayed on a single scaffold high affinityantibodies were generated. For further details of strategies and methodsfor generating synthetic antibody libraries with single template, see,e.g., WO 2005/012359 published Feb. 10, 2005, the entire disclosure ofwhich is expressly incorporated herein by reference.

Solution phase panning with naïve libraries was performed againstbiotinylated murine VEGF in solution and then captured by 5 ug/mlneutravidin immobilized on MaxiSorp™ immunoplates. After three rounds ofselection with decreasing concentration of biotinylated murine VEGF,clones were randomly picked and specific binders were identified usingphage ELISA. For each positive phage clone, variable regions of heavyand light chains were subcloned into pRK expression vectors that wereengineered to express full-length IgG chains. Heavy chain and lightchain constructs were co-transfected into 293 or CHO cells, and theexpressed antibodies were purified from serum-free medium using proteinA affinity column. For affinity maturation, phage libraries withdifferent combination of CDR loops (CDR-H1 and H2, CDR-L1, L2 and L3)derived from the initial clone of interest were constructed by softrandomization strategy so that each selected position was mutated to anon-wild type residue or maintained as wild type at about 50:50frequency (Lee, C. V et al., Blood, 108:3103-3111, 2006). High affinityclones were then identified through four rounds of solution phasepanning against biotinylated human VEGF as described. Decreasingbiotinylated antigen concentration allowed more stringency in panning.

Example 2 Anti-VEGF Antibodies Binding Affinities

To determine binding affinities of anti-VEGF IgGs, surface plasmonresonance (SRP) measurement with a BIAcore™-3000 instrument was used.Carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.) wereactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Human or murine VEGF was immobilized to achieveapproximately 60 response units (RU) of coupled protein. See FIG. 10.For kinetics measurements, two-fold serial dilutions of anti-VEGF IgG(7.8 nM to 500 nM) were injected separately in PBT buffer (PBS with0.05% (v/v) Tween 20) at 37° C. with a flow rate of 25 μl/min.Association rates (k_(on)) and dissociation rates (k_(off)) werecalculated using a bivalent binding model (BIAcore Evaluation Softwareversion 3.2). The equilibrium dissociation constant (K_(D)) wascalculated as the ratio k_(off)/k_(on).

Due to the unmeaureable off-rate of B20.4.1, IgGs were immobilized toachieve approximately 1000 response units (RU) of coupled protein. SeeFIG. 11. Two-fold serial dilutions of human or murine VEGF (7.8 nM to500 nM) were injected in PBT buffer (PBS with 0.05% (v/v) Tween 20) at37° C. with a flow rate of 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) were calculated using a simple one to oneLangmuir binding model (BIAcore Evaluation Software version 3.2). toobtain the equilibrium dissociation constant (K_(D)).

Example 3 HUVEC Thymidine Incorporation Assay

To study the function of anti-VEGF antibodies by inhibiting VEGF-inducedcell proliferation, HUVEC thymidine incorporation assay was performed.Human umbilical vein endothelial cells (HUVEC) (Clontech, Mountain View,Calif.) were grown and assayed as described. Approximately 3000 HUVECswere plated in each well of the 96-well cell culture plate and incubatedin Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplementedwith 1.5% (v/v) fetal bovine serum (assay medium) for 18 hours. Freshassay medium with fixed amounts of human VEGF (0.1 nM finalconcentration), determined by first titrating VEGF that can stimulatesubmaximal DNA synthesis, and increasing concentrations of anti-VEGFIgGs were then added to the cells. After incubation at 37° C. for 18hours, cells were pulsed with 0.5 μCi/well of [³H] thymidine for 24hours and harvested for counting with TopCount Microplate Scintillationcounter as described.

The HUVEC thymidine incorporation assay shows that B20 variants caneffectively inhibit the HUVEC cell proliferation. FIG. 12 shows thatB20-4.1.1 and B20-4.1.1RR have similar inhibition as G6-31.

Example 4 Endothelial Cell Proliferation Assay

To determine binding specificity and blocking activity of B20-4.1.1, acell-based assay using bovine retinal microvascular endothelial cells(BRMEs) was performed, wherein the antibody was tested for its abilityto block human or murine VEGF-induced cell proliferation. BRMEs wereseeded at a density of 500 cells/well in 96-well plates in growth medium(low-glucose DMEM supplemented with 10% calf serum, 2 mM glutamine, andantibiotics). For the inhibition assay, B20-4.1.1 was added at theindicated concentration (ng/ml) to triplicate wells (FIG. 13). After 0.5hours, human VEGF-A (hVEGF) or mouse VEGF-A (mVEGF) was added to a finalconcentration of 6 ng/ml. After six to seven days, cell growth wasdetermined by Alamar blue (BioSource; Invitrogen). Fluorescence wasmonitored at 530 nm excitation wavelength and 590 nm emissionwavelength.

As shown in FIG. 13, B20-4.1.1 reduced the ability of both hVEGF andmVEGF to promote BRME proliferation. As shown in FIG. 16, the avastinantibody reduced the ability of hVEGF to promote BRME proliferation. Theavastin antibody did not inhibit mVEGF-induced proliferation asinhibition of mVEGF was not observed at a concentration of up to 1500 nMof the avastin antibody.

Example 5 Tumor Inhibition In Vivo Studies

A549 cells (human lung cancer cells) and MDA-MB231 cells (human breastcancer cells) were grown in cell culture and were injectedsubcutaneously into 8- to 12-week-old beige nude mice at a cell densityof ˜5×10⁶ cells/mouse. Forty-eight hours after tumor cell inoculation orwhen the tumor reached approximately 200 mm³ in size, mice (n=10) wereinjected intraperitoneally with B20-4.1.1 at a concentration of 5 mg/kgor with vehicle buffer. Antibodies were administered twice weeklythereafter. Tumor volumes were measured with calipers at the indicatedtime points.

As shown in FIGS. 14 and 15, B20-4.1.1 was effective in reducing tumorvolumes in mice injected with either A549 cells (FIG. 14) or MDA-MB231cells (FIG. 15).

Although in the foregoing description the invention is illustrated withreference to certain embodiments, it is not so limited. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed:
 1. A method for blocking or reducing relapse tumorgrowth or relapse cancer cell growth comprising administering aneffective amount of the anti-VEGF antibody to a subject in need of suchtreatment, wherein the anti-VEGF antibody comprises: (a) an HVR-L1comprising the amino acid sequence of X₁X₂R X₃SL wherein the HVR-L1comprises 1, 2 or 3 substitutions in any combination of the followingpositions: X₁ is G or A; X₂ is V or and/or X₃ is T or R; (b) an HVR-L2comprising the amino acid sequence of DASSLA (SEQ ID NO: 6); (c) anHVR-L3 comprising the amino acid sequence of SYKSPL (SEQ ID NO: 7); (d)an HVR-H1 comprising the amino acid sequence of SISGSWIF (SEQ ID NO: 1);(e) an HVR-H2 comprising the amino acid sequence of GAIWPFGGYTH (SEQ IDNO: 2); and (f) an HVR-H3 comprising the amino acid sequence ofRWGHSTSPWAMDY (SEQ ID NO: 3).
 2. The method of claim 1, comprisingadministering an effective amount of an anti-VEGF antibody to a subjectin need of such treatment, wherein the anti-VEGF antibody comprises: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 4; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 6; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:
 7. 3. The method ofclaim 1, comprising administering an effective amount of an anti-VEGFantibody to a subject in need of such treatment, wherein the anti-VEGFantibody comprises: (a) an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 1; (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 2; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3;(d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 5; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 6; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO:
 7. 4. The methodof claim 1, wherein the antibody is a monoclonal, humanized, or humanantibody.
 5. The method of claim 1, wherein the antibody is an antibodyfragment.
 6. The method of claim 1, wherein the subject is human.
 7. Amethod for blocking or reducing relapse tumor growth or relapse cancercell growth, comprising administering an effective amount of ananti-VEGF antibody to a subject in need of such treatment, wherein theanti-VEGF antibody comprises a heavy chain variable domain comprising anamino acid sequence of SEQ ID NO: 43 and a light chain variable domaincomprising an amino acid sequence of SEQ ID NO: 44 or SEQ ID NO:
 45. 8.The method of claim 7, wherein the heavy chain variable domain comprisesthe amino acid sequence of SEQ ID NO: 43 and the light chain variabledomain comprises the amino acid sequence of SEQ ID NO:
 44. 9. The methodof claim 7, wherein the heavy chain variable domain comprises the aminoacid sequence of SEQ ID NO: 43 and the light chain variable domaincomprises the amino acid sequence of SEQ ID NO:
 45. 10. The method ofclaim 7, wherein the antibody is a monoclonal, humanized, or humanantibody.
 11. The method of claim 7, wherein the antibody is an antibodyfragment.
 12. The method of claim 7, wherein the subject is human.